The ability to modulate the adaptive immune response in patients offers the potential for powerful targeted therapies with improved safety compared with currently available conventional drugs. Methods aimed at either boosting or suppressing T cell responses could be successfully exploited as therapies in a number of diseases. This is because T cells form an important component of the adaptive immune system mediating both specificity and memory for a pathogenic challenge, providing a focus for developing highly selective therapies to replace current blanket therapies that affect the immune system as a whole and control rather than cure disease.
Full activation of T cells requires stimulation through the T cell antigen receptor and additional signalling via co-stimulatory molecules displayed on the cell surface of T cells, primarily the CD28 receptor (1-3). The ligands for CD28 are CD80 (B7.1) and CD86 (B7.2), displayed by cells such as dendritic cells, macrophages and B cells that also present antigen to the receptive T cell (4,5). Engagement of CD28 by CD80 or CD86 stimulates signalling pathways that stabilise and amplify the antigen-specific T cell response. This is characterised by increased T cell production of the cytokine IL-2, expression of proteins that suppress apoptosis (Bcl-XL), and secretion of effector cytokines that amplify the antigen-specific immune response.
CTLA-4 is a structural homologue of CD28, both are members of the immunoglobulin superfamily, share approximately 30% amino acid sequence homology, and in humans, are located in the same region of chromosome 2 (6-8). Notably, both retain sequence motifs important for binding CD80/CD86. However, CTLA-4 is widely accepted as a receptor with opposing effects on T cell activity compared with CD28, delivering inhibitory rather than stimulatory signals to activated T cells. It is generally acknowledged to be a counter-receptor that can attenuate the intensity of the immune response prosecuted by the activated T cell on which it is displayed (9,10). It is also widely accepted that CD4+ regulatory T cells constitutively express the molecule on their cell-surface, whereas other effector T cell subsets e.g., CD4+ Th1 T cells, only express it following activation (11-13). There is further evidence that the molecule participates in Treg function and thus CTLA-4 may be involved in regulating the immune response both by modulating the intrinsic activity of the cell that expresses it and by inhibiting other activated T cells during an immune response (14-18).
Attempts to delineate the role of CTLA-4 in T cell stimulation demonstrated that it is important as an inhibitory regulator of T cells. First, mice deficient for the CTLA-4 gene die 3-5 weeks after birth from a massive lymphoproliferative disorder in which activated T cell blasts accumulate rapidly in lymphatic tissues and progress to infiltrate other organs and tissues of the body (19,20). This provides evidence that CTLA-4 has a role both in limiting the activation status of T cells and maintaining T cell homeostasis. Further, studies with antibodies specific for CTLA-4 have been used to evaluate its role in purified T cell populations and found that antibody cross-linking of CTLA-4 on the cell surface inhibits T cell proliferation and IL-2 production (21-24). These effects directly opposed the stimulatory effects mediated by CD28 and so it is likely that the CD28 and CTLA-4 co-stimulation molecules combine to modulate T cell antigen receptor stimulation by delivering stimulatory and inhibitory signals respectively.
Antibody blockade of CTLA-4 has been widely used to demonstrate that inhibition of CTLA-4 function enhances T cell activity in a range of disease situations, including cancer, infection and other immune-related scenarios. In cancer, antibody blockade of CTLA-4 function has been established as a potentially viable method of establishing powerful anti-tumour T cell responses (25-31; see also U.S. Pat. No. 6,984,720 assigned to Medarex, Inc.). The first experiments were conducted in murine models of cancer. Blockade of CTLA-4 enhanced anti-tumor T cell immune responses leading to successful reduction and abolition of tumours. Blockade of CTLA-4 has been performed in cancer models using antibody alone or in combination with a vaccine specific to the cancer. It seems that the natural immunogenicity of the particular tumour is a determining factor of whether CTLA-4 blockade alone, or blockade in combination with a vaccine or other immune activator is sufficient to generate a successful anti-tumour immune response. Initial studies of CTLA-4 blockade in murine models of cancer have led to similar studies in humans and at least two monoclonal antibodies specific for human CTLA-4 have been extensively studied in clinical trials aimed at treating a diverse range of cancers (31).
In connection with infection, antibody blockade of CTLA-4 function demonstrated greatly enhanced immune responses including anti-parasitic, anti-bacterial and anti-viral responses enhancing a spectrum of immunity including increased antigen specific antibody, and Th1/Th2 T helper cell responses (32-36). Antibody blockade of CTLA-4 also enhances autoimmune responses (37).
Most research concerning CTLA-4 has focussed on the receptor form of the molecule but there are alternative genetic isoforms, which in protein form do not reside on the cell surface of T cells (reviewed by Teft et al. (2006) (38)).
The full length membrane-bound isoform of CTLA-4 is encoded in humans by four exons (1-4) on chromosome 2, but there are other mRNA transcripts including one that generates a secretable soluble form of CTLA-4 (sCTLA-4) (39,40). This alternatively spliced transcript is missing exon 3, corresponding to the transmembrane domain of full-length CTLA-4, and a reading frame shift of exon 4 replaces the cytoplasmic tail sequence with a different C-terminal amino acid sequence of no known function. Like full-length CTLA-4, sCTLA-4 has the capacity to bind B7.1/B7.2 co-stimulator ligands on APC but its role as a regulator of antigen-specific immune responses has not been evaluated. Initial studies indicated that resting T cells are the primary source of sCTLA-4, which after non-specific activation with anti-CD3 mAb, rapidly switch to producing the full-length isoform to regulate the immune response.
The Oaks and Hallett (43) describe the production of a rabbit polyclonal antiserum to the C terminal region of sCTLA-4. The antiserum was used in Western blots to detect presence of the sCTLA-4 protein. It was not used in any functional assays.
Single nucleotide polymorphisms (SNP) within the CTLA-4 gene locus have been associated with susceptibility for autoimmune disease. A powerful population analysis of a CTLA-4 associated SNP (CT60) found that a particular haplotype (homozygous g/g) correlated with increased susceptibility for Graves' disease, autoimmune hypothyroidism and type 1 diabetes (41). The SNP is located downstream of the 4 CTLA-4 encoding exons and subsequent analysis indicated that the susceptibility SNP is influential upon CTLA-4 by determining a relative decrease in the amount of sCTLA-4 protein produced. Expression levels of full length CTLA-4 were not affected. These data provided evidence that sCTLA-4 may in fact have a role in regulating the immune system.
WO2005/072340 describes variants of the CTLA-4 receptor and soluble CTLA-4 molecules.
Other CTLA-4 alternative isoforms include IiCTLA-4, present in rodents but not humans, where the alternative transcript lacks exon 2, and another encoded only by the exons 1 and 4 (38). This latter transcript, present in humans, has no reported function at present.